SUMMARY Intracellular cargo transport, such as insulin granules destined for secretion in pancreatic ?-cells, relies on myosin Va (myoVa) molecular motors. This double-headed molecular motor carries its cargo by stepping processively for considerable distances along actin tracks. To successfully deliver cargo, a team of myoVa motors must overcome the physical challenges presented by the 3-dimensional (3D) complex networks of branched and bundled actin filaments that comprise the actin cytoskeleton. To determine how efficient myoVa cargo transport and delivery are accomplished despite the physical challenges presented by the cell's multitude of actin structures and complex networks, we will develop complex but well-defined, 3-dimensional (3D) actin networks formed in vitro using multifunctional actin-binding proteins, and; 2) create structurally- defined ?designer? actin networks inside cultured cells. By using state-of-the-art single molecule biophysical techniques with high spatial (6nm) and temporal (10ms) resolution, we will define how myoVa motor teams respond to these physical challenges. To begin simulating the complex 3D intracellular cytoskeleton, in Aim #1, we will suspend actin filaments to create actin filament intersections in 3D. Similarly, branched actin filaments formed by Arp2/3 will also be suspended. These 3D actin filament intersections and branch points will then challenge a team of myoVa motors carrying a lipid-bound, liposome cargo. Knowing the precise spatial relation between the cargo and the actin tracks using super-resolution STORM imaging, and by varying the number of motors and the fluidity of the cargo's lipid coating, we will provide a mechanistic basis for the directional outcome as the cargo and its team of motors maneuvers through the intersection or branch point. These studies will inform the studies in Aim #2 in which we will create structurally and mechanically defined 3D actin networks of branched and bundled actin filaments as models of the cell's complex actin cytoskeleton. The cellular structures to be modeled are the actin cortex with its Arp2/3 branched filaments and filopodia and stress fibers that are parallel actin bundles formed by fascin and ?-actinin cross-linkers, respectively. We propose that a functional interplay exists between the properties of the actin tracks, motors, and cargos, which determines how teams of myoVa motors meet the cellular demands placed on them by this multitude of actin structures. The data obtained will provide a rich, mechano-spatial knowledgebase for the field and serve as a foundation for understanding myoVa transport in the complex architectural environment of the cell, which we will control by culturing cells on patterned substrates so as to create ?designer? actin cytoskeletons. Thus, the proposed studies will provide a mechanistic understanding of how efficient myoVa transport system is designed for delivery and retention of cargo at its destination, such as insulin granules.